ES2654336T3 - Factor VIII, von Willebrand factor or its complexes with prolonged in vivo half-life - Google Patents

Abstract

A modified factor VIII or a complex comprising modified factor VIII and unmodified VWF, wherein the modified factor VIII is fused in a C-terminal part of the primary translation polypeptide of factor VIII with the N-terminal part of an albumin.

Description

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DESCRIPTION

Factor VIII, von Willebrand factor or its complexes with prolonged in vivo half-life Field of the invention:

The present invention relates to modified nucleic acid sequences encoding the coagulation factor VIII (FVIII) and von Willebrand factor (VWF, by its acronym) unmodified, as well as their complexes and derivatives thereof, to recombinant expression vectors containing such nucleic acid sequences, host cells transformed with such recombinant expression vectors, recombinant polypeptides and derivatives encoded by said nucleic acid sequences for recombinant and derived polypeptides have biological activities together with an in vivo half-life prolonged and / or improved in vivo recovery compared to unmodified naturally occurring protein. The invention also relates to corresponding FVIII sequences that result in improved performance of the expression. The present invention further relates to processes for the preparation of such recombinant proteins and their derivatives. The invention also relates to a transfer vector for use in human gene therapy, which comprises such modified nucleic acid sequences.

Background of the invention:

Several bleeding disorders caused by deficiencies of clotting factors in the blood. The most common disorders are hemophilia A and B, which are the result of deficiencies of coagulation factor VIII blood and IX, respectively. Another known bleeding disorder is von Willebrand disease.

In plasma, FVIII exists mostly as a noncovalent complex with VWF and its coagulant function is to accelerate the conversion, dependent factor IXa, factor X to Xa. Due to the formation of FVIII and VWF complex, long it assumed the functions of FVIII and VWF were two functions on the same molecule. Only in the 70s it became obvious that FVIII and VWF were different molecules forming a complex under physiological conditions. Then, in the 80s, the dissociation constant of about 0.2 nmol / L (. Leyte et al, Biochem J 1989, 257: 679-683) was determined and the DNA sequence of both molecules was studied.

Classic hemophilia or hemophilia A is an inherited bleeding disorder. It is the result of a deficiency related to the X chromosome FVIII clotting blood and affects almost exclusively males with an incidence of between one and two individuals per 10 000. The defect on the X chromosome is transmitted by carriers Females who are not hemophiliacs per se. The clinical manifestation of hemophilia A is an increased bleeding tendency. Before treatment with FVIII concentrates was introduced, the average life expectancy for a person with severe hemophilia was less than 20 years. The use of FVIII concentrates from plasma has considerably improved the situation for patients with hemophilia A, increasing the average life expectancy considerably and giving most of them the possibility to live a more or less normal life. However, there have been some problems with the plasma derived concentrates and their use, the most serious of which have been the transmission of viruses. So far, the viruses that cause hepatitis B, hepatitis other than type A or B and AIDS have wreaked havoc on the population. Since then, recently they developed different methods of virus inactivation and new highly purified factor VIII concentrates, which set a very high safety standard also for plasma-derived FVIII.

CDNA cloning for FVIII (Wood et al 1984 Nature 312: 330-336; Vehar et al 1984. Nature 312:.. 337-342) allowed to express FVIII recombinantly, leading to the development of several products of FVIII recombinants, which were approved by the regulatory authorities between 1992 and 2003. the fact that the domain B core of the polypeptide chain of FVIII residing between Arg-740 and Glu-1649 amino acids seems not to be necessary for biological activity complete has also led to the development of an FVIII with the deleted B domain.

The mature FVIII molecule consists of 2332 amino acids, which can be grouped into three domains A homologues, homologues two domains C and a B domain, which are arranged in the following order: A1-A2 B-A3-C1- C2 The complete amino acid sequence of mature human FVIII is shown in SEQ ID NO: 15. During its secretion into plasma FVIII it is processed intracellularly to obtain a series of heterodimers bound metal ions as FVIII single strand is cleaved at the border of B-A3 at different locations of the domain B. This processing provides heterogeneous chain molecules heavy domain consisting of the A1, A2 and various parts of the B domain, which have a molecular size of between 90 kDa and 200 kDa. The heavy chains are linked by a metal ion to the light chains, which are constituted by domain A3, C1 and C2 (Saenko et al. 2002. Vox Sang. 83: 89-96). In plasma, this heterodimeric FVIII binds

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with high affinity to von Willebrand factor (VWF), which protects against premature metabolism. FVIII half-life activated not bound VWF is about 12 hours in plasma.

The FVIII coagulation factor activated by proteolytic cleavage by FXa and thrombin at Arg372 and Arg740 amino acid heavy chain and at Arg1689 in the light chain, which triggers the release of von Willebrand factor and generation heterotrimer FVIII activated, which will form the tenase complex on phospholipid surfaces with FIXa and FX provided that Ca2 + present there. The heterotrimer consists of the A1 domain, a fragment of 50 kDa A2 domain, a fragment of 43 kDa, and the light chain (A3-C1-C2), a fragment of 73 kDa. Thus, the active form of FVIII (FVIIIa) consists of an A1-subunit associated through binding to a divalent metal ion to an A3-C1-C2 light chain cleaved by thrombin and a free A2 subunit associated form relatively weak with domain A1 and A3.

To prevent excessive clotting FVIII must be deactivated shortly after activation. The deactivation of FVIIIa by activated Protein C (APC) by cleavage in Arg336 and Arg562 is not considered to be an important speed limiting step. Instead, it is believed to be the dissociation of the A2 subunit covalently bound the heterotrimer which is the speed limiting step in inactivating FVIIIa after thrombin activation (Fay et al. 1991. J. Biol. Chem 266 8957, Fay and Smudzin 1992. J. Biol. Chem. 267: 13246-50). This is a quick process, which explains the short half-life of FVIIIa in plasma, which is only 2.1 minutes (Saenko et al. 2002. Vox Sang. 83: 89-96).

In patients with severe haemophilia undergoing prophylactic treatment FVIII has to be administered intravenously (i.v.) about 3 times per week due to the short half-life in plasma FVIII of about 12 to 14 hours. Each administration i.v. It is annoying, it is associated with pain and carries the risk of infection, especially since it is mainly performed at home by the same patients or parents of children who have been diagnosed with hemophilia A.

Therefore, it would be highly desirable to create an FVIII with a higher functional half-life, which would allow the development of pharmaceutical compositions containing FVIII, which should be administered less frequently.

There have been several attempts to prolong the half-life of FVIII not activated, either by reducing its interaction with (WO 03 / 093313A2, WO 02 / 060951A2) cellular receptors, covalently attaching polymers to FVIII (WO 94/15625, WO 97/11957 and US 4970300), encapsulating FVIII (WO 99/55306), introducing novel binding sites metals (WO 97/03193), by covalently attaching the A2 domain to the A3 domain either by peptidic linkage (WO 97/40145 and WO 03 / 087,355) or disulfide (WO 02 / 103024A2) or by covalently attaching the A1 domain to the A2 domain (WO2006 / 108590).

Another strategy to improve the functional half-life of FVIII or VWF is PEGylation of FVIII (WO 2007/126808, WO 2006/053299, WO 2004/075923) or PEGylation of VWF (WO 2006/071801), where the pegylated VWF, having a greater half-life, indirectly also enhance the half-life of FVIII present in plasma.

Since none of the strategies described above resulted in still a pharmaceutical agent of FVIII approved and because the introduction of mutations in the naturally occurring sequence of FVIII or introducing chemical modifications entails at least a theoretical risk that immunogenic variants FVIII believe, still need to develop modified coagulation factor molecules VIII exhibiting a long half-life.

Given the potential thrombogenic risk it is more desirable to prolong the half life of non-activated form of FVIII than that of FVIIIa.

The VWF factor, which is absent, functionally defective or only available at a reduced various forms of von Willebrand disease (VWD, for its acronym in English) amount is a multimeric adhesive glycoprotein present in plasma of mammals , which presents multiple physiological functions. During primary hemostasis, VWF acts as a mediator between specific receptors on the platelet surface and components of the extracellular matrix such as collagen. Moreover, VWF serves as a carrier and stabilizing protein for procoagulant FVIII. Factor VWF is synthesized in endothelial cells and megakaryocytes as a 2813 amino acid precursor molecule. The amino acid sequence and the cDNA sequence of naturally occurring VWF are disclosed in Collins et al. 1987, Proc Natl. Acad. Sci. USA 84: 4393-4397. The precursor polypeptide, pre-pro-VWF, consists of a signal peptide of 22 residues, a propeptide of 741 residues and polypeptide 2,050 residues found in VWF in mature plasma (Fischer et al, FEBS Lett 351..: 345-348, 1994). After cleavage of the signal peptide in the endoplasmatic reticulum a C-terminal disulfide bridge is formed between two monomers of VWF. During further transport through the secretory pathway 12 carbohydrate side chains attached to N and 10 O. are added together to Notably VWF dimers disulfide bridges multimerized by N-terminal propeptide and the amino acid 741 length is cleaved by the enzyme PACE / furin in the late Golgi apparatus. The propeptide, as well as

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VWF high molecular weight multimers (VWF-HMWM) are stored in Weibel-Pallade bodies of endothelial cells or in Granules a of platelets.

Once secreted in plasma, the ADAMTS13 protease cleaves VWF in the A1 domain of VWF. Therefore, VWF in plasma consists of a full range of multimers ranging from individual 500 kDa dimers to multimers consisting of more than 20 dimers of a molecular weight greater than 10,000 kDa. Herein, VWF HMWM-wraps powerful hemostatic activity, which can be measured according to ristocetin cofactor activity (VWF: RCo). The higher the ratio of VWF: RCo / VWF antigen, the higher the relative amount of high molecular weight multimers.

Defects in VWF cause von Willebrand disease (VWD), which is characterized by a more or less pronounced bleeding phenotype. VWD type 3 is the most severe form in which VWF is completely absent; Type 1 VWD is related to a quantitative loss of VWF and its phenotype can be very slight. VWD type 2 relates to qualitative defects are VWF and can be as severe as VWD type 3. VWD type 2 has many sub-forms, some of which are associated with the loss or reduction of molecular weight multimers high. VWD type 2a is characterized by a loss of multimers large and intermediate. VWD type 2B is characterized by a loss of multimers of higher molecular weight.

VWD is the most common inherited bleeding disorder in humans and can be treated by replacement therapy with concentrates containing VWF of plasma or recombinant origin. The VWF factor can be prepared from human plasma as described, for example in EP 05503991. EP 0784632 describes a method for isolating recombinant VWF.

In plasma, FVIII binds with high affinity to VWF, which protects it from premature catabolism and thus, in addition to its role in primary hemostasis, plays a crucial role in regulating the plasma levels of FVIII and, as a consequence, it is also a central factor in controlling secondary hemostasis. FVIII half-life activated not bound VWF is about 12 to 14 hours in plasma. In von Willebrand disease type 3, where not there or almost no VWF present, the half-life of FVIII is only about 6 hours, resulting in symptoms of Hemophilia A mild to moderate in these patients, since to the reduction of FVIII concentrations. The stabilizing effect of VWF on FVIII has also been used to facilitate recombinant expression of FVIII in CHO (Kaufman et al. 1989, Mol Cell Biol) cells.

Until today the standard treatment of Hemophilia A and VWD involves frequent intravenous infusions of preparations of FVIII and VWF concentrates or of concentrates comprising a complex of FVIII and VWF derived from plasmas of human donors or in the case of FVIII, of pharmaceutical preparations based on recombinant FVIII. Although these replacement therapies are generally effective, e.g. g., in patients with severe haemophilia undergoing prophylactic treatment FVIII must be administered intravenously (i.v.) about 3 times per week due to the short plasma half life of FVIII of about 12 hours. Already above levels of 1% of FVIII activity in nonhemophilic people, p. eg., an increase FVIII levels of 0.01 U / mL, severe hemophilia A hemophilia becomes moderate. In prophylactic therapy, dosage regimens are designed so that the background level of FVIII activity is not reduced below levels of 2-3% of the FVIII activity in non-hemophiliacs. Each administration i.v. is annoying, is associated with pain and carries the risk of infection, especially because it is mainly carried out in a treatment at home by the same patients or parents of children who have been diagnosed with hemophilia A. In addition, iv injections frequent inevitably cause the formation of scars, which interferes with future infusions. Because prophylactic treatment in severe hemophilia starts early in life, often having children less than 2 years of age, it is even more difficult to inject FVIII 3 times a week in the veins of such small patients. For a limited period, the implantation of port systems may offer an alternative. Despite the fact that repeated infections may occur and that can cause problems ports during exercise, usually considered favorable nevertheless compared to intravenous injections.

In the prior art, they described fusions of coagulation factors to albumin (WO 01/79271), alpha-fetoprotein (WO 2005/024044) and immunoglobulin (WO 2004/101740) as half-life enhancing polypeptides. It was believed that these were attached to the carboxyl or amino terminus or to both ends of the respective therapeutic protein moiety, occasionally connected by peptide linkers, preferably by connectors consisting of glycine and serine.

Ballance et al. (WO 01/79271) described N-terminal or C-terminal fusion polypeptides of a multitude of different therapeutic polypeptides fused with human serum albumin. exhaustive lists of possible components for fusion without describing experimental data for almost any of these polypeptides, whether the respective fusion proteins with albumin actually retain biological activity and have improved properties or not described. Among said list of therapeutic polypeptides, FVIII and VWF are also mentioned.

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One skilled in the art would not seriously considered a C-terminal fusion as the C2 domain of FVIII at the C-terminus of FVIII part between amino acid 2303 and 2332 of FVIII comprises a binding site for platelet membrane , which is essential for the function of FVIII. This is why there are many amino acid mutations known in this region which cause haemophilia A. Therefore, it was surprising that a heterologous polypeptide relatively large as albumin could be fused at the C-terminus of FVIII without suppress the function of FVIII by suppressing its binding to platelets. In addition, the c2 domain also contains a binding site for VWF. This site together with the amino acid sequence 16491689, is responsible for the high affinity binding of FVIII to VWF. Accordingly, one skilled in the art would also not have expected that a FVIII albumin fusion would retain its C-terminal binding VWF.

Surprisingly, it was observed that, contrary to the prediction of Ballance et al., A fusion of albumin at the N-terminal end of FVIII was not secreted into the culture medium. Therefore and because of the above-listed reasons, has now been found, even more surprisingly, a FVIII fused at its C-terminal albumin is secreted into the culture medium and retains its biological function including binding to platelet membrane already activated VWF.

It was also found, surprisingly, that the modified FVIII of the invention exhibits an increased in vivo recovery of about 20% compared to naturally occurring FVIII.

Description of the invention

An object of this invention to provide a modified FVIII or modified FVIII complex with VWF unmodified present an improved in vivo half-life.

The term "modified FVIII" in the sense of the invention relates to polypeptides which are fused FVIII polypeptides which enhance half-life, which also encompass natural alleles, variants, deletions and insertions of FVIII.

Another object of this invention to provide a modified FVIII, as well as complexes of modified FVIII with modified VWF which have not improved in vivo recovery.

Another object of the invention is that this modified FVIII or modified FVIII complex with VWF unmodified, can be expressed by mammalian cells and retain their respective biological activities.

In summary, surprisingly the modified FVIII or FVIII complexes modified with unmodified VWF of the invention, have conserved biological activity and have increased half-life in vivo and recovery in vivo.

A possible additional benefit of those embodiments of the present invention in which modified FVIII and in which the A2 domain remains attached only noncovalently the A3 domain after activation is that only increases the half-life of the form not activated FVIII, while the half-life of the activated form of FVIII remains essentially the same, which can result in a reduced risk of thrombogenicity as compared to variants of FVIII leading to stabilization of the form FVIII activated.

The modified FVIII or modified FVIII complex with VWF unmodified of the invention can be generated by fusing a moiety of a protein that enhances half-life (HLEP, by its acronym) with the C-terminal FVIII.

HLEPs, within the meaning of the present invention, are selected from a group consisting of members of human albumin.

Accordingly, the present invention relates to a modified or FVIII FVIII modified VWF unmodified containing the C-terminal part of the modified FVIII a fusion with a HLEP, characterized by the FVIII modified or complex FVIII Modified with unmodified VWF has a prolonged functional half-life compared to the functional half-life of FVIII of natural origin or of the VWF complex of natural origin and FVIII of natural origin.

The present invention also relates to C-terminal fusions to more than one HLEP wherein the HLEP, which is fused several times.

The present invention also relates to a modified FVIII containing the C-terminal part a fusion with a HLEP, it characterized in that the modified FVIII or the complex of modified FVIII with VWF unmodified presents an in vivo recovery improved compared with the in vivo recovery of FVIII of natural origin or the VWF complex of natural origin and FVIII of natural origin.

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Another embodiment of the invention are polypeptides modified FVIII containing the C-terminal part a fusion with a HLEP, characterized in that the modified FVIII is secreted into a fermentation medium with greater than a FVIII naturally occurring performance.

Another aspect of the invention consists of polynucleotides or combinations of polynucleotides encoding the modified VWF.

The invention further relates to plasmids or vectors comprising a polynucleotide described herein and to host cells comprising a polynucleotide or a plasmid or vector as described herein.

Another aspect of the invention consists of a method for producing a modified FVIII or a modified FVIII complex with unmodified VWF comprising:

(A) culturing host cells of the invention under conditions such that the modified coagulation factor is expressed; Y

(b) optionally recovering the modified coagulation factor from the host cells or from the culture medium.

The invention further relates to pharmaceutical compositions comprising a modified FVIII or a modified FVIII complex with unmodified VWF, a polynucleotide or a plasmid or vector described herein.

Another aspect of the invention is the use of a modified FVIII or a complex of modified FVIII VWF unmodified, one or more polynucleotides, or one or more plasmids or vectors, or of host cells according to this invention for the development of a medication for the treatment or prevention of a blood clotting disorder.

Detailed description of the invention

The invention relates to a complex comprising FVIII and VWF or one of its individual polypeptidic components, where the FVIII of said complex is fused at the C-terminal part of its primary translation product to the N-terminus of a polypeptide improves half-life (HLEP).

The invention also relates to a modified FVIII or a complex comprising modified FVIII and non-modified, wherein the modified FVIII is fused at a C-terminal part of the primary translation polypeptide of FVIII to the N-terminal part a HLEP.

In preferred embodiments, the invention relates to a modified FVIII or a complex comprising modified and unmodified FVIII, wherein

(a) the modified FVIII exhibits a prolonged functional half-life compared to the functional half-life of naturally occurring FVIII or

(b) the complex comprising modified FVIII and unmodified FVIII has a prolonged functional half-life compared to the functional half-life of the corresponding complex comprising naturally occurring FVIII and naturally occurring VWF.

A preferred embodiment of the invention is a modified polypeptide or a complex comprising said modified polypeptide or a complex comprising said modified polypeptides as described above, wherein the modified polypeptide has an increased functional half life of at least 25% compared to the complex comprising said modified polypeptide or a complex comprising said modified polypeptides has an increased functional half life of at least 25% compared to the corresponding complex of FVIII functional half-life of the corresponding naturally occurring polypeptide or Natural origin and VWF of natural origin.

Another embodiment of the invention consists of a modified FVIII or a complex comprising modified FVIII and unmodified VWF, wherein

(A) the modified FVIII has a prolonged antigen half-life compared to the half life of FVIII antigen naturally occurring or

(b) the complex comprising modified FVIII and unmodified VWF has a prolonged half-life of the antigen compared to the half-life of the corresponding complex antigen comprising FVIII of natural origin and VWF of natural origin.

A preferred embodiment of the invention is a modified polypeptide or a complex comprising said modified polypeptide or a complex comprising said modified polypeptides as described

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above, wherein the modified polypeptide has an increased half-life of the antigen of at least 25% compared to the half life of the antigen of the corresponding naturally occurring polypeptide or the complex comprising said modified polypeptide or a complex comprising said modified polypeptides has increased half-life of the antigen of at least 25% compared to the corresponding complex of FVIII and VWF naturally occurring naturally occurring.

A further embodiment of the invention consists of a modified FVIII or a complex comprising modified FVIII and unmodified VWF, where

(a) the modified FVIII exhibits an increase in in vivo recovery compared to in vivo recovery of naturally occurring FVIII or

(b) the complex comprising modified FVIII and unmodified VWF exhibits an increase in in vivo recovery compared to the in vivo recovery of the corresponding complex comprising naturally occurring FVIII and VWF of natural origin.

Another preferred embodiment of the invention consists of a modified polypeptide or a complex comprising said modified polypeptide or a complex comprising said modified polypeptides as described above, wherein the modified polypeptide exhibits an increase in in vivo recovery of at least 10 % in comparison to the in vivo recovery of the corresponding naturally occurring polypeptide or the complex comprising said modified polypeptide or a complex comprising said modified polypeptides exhibits an increase in in vivo recovery of at least 10% compared to the corresponding complex of FVIII of natural origin and vWf of natural origin.

Another preferred embodiment of the invention consists of

(a) a modified polypeptide or a complex comprising said modified polypeptide or a complex comprising said modified polypeptides as described above, where at least one polypeptide component of said complex is fused into the C-terminal amino acid of its translation product primary with the N-terminal part of an HLEP, or

(b) a modified polypeptide or a complex comprising said modified polypeptide or a complex comprising said modified polypeptides as described above, where at least one polypeptide component of said complex is fused in the C-terminal part of its translation product primary with the N-terminal amino acid of an HLEP, or

(c) a modified polypeptide or a complex comprising said modified polypeptide or a complex comprising said modified polypeptides as described above, where at least one polypeptide component of said complex is fused into the C-terminal amino acid of its translation product primary with the N-terminal amino acid of an HLEP.

Another preferred embodiment of the invention consists of a modified polypeptide or a complex comprising said modified polypeptide or a complex comprising said modified polypeptides as described above, wherein the modified polypeptide has at least 10% of the biological activity of the polypeptide of Natural origin or the complex comprising the modified polypeptide or a complex comprising said modified polypeptides has at least 10% of the biological activity of the corresponding complex of naturally occurring FVIII and naturally occurring VWF.

The present invention also encompasses a method for preparing a modified with a greater functional half-life, which comprises fusing the N-terminal part of a polypeptide that improves the half-life with a C-terminal part of the primary translation polypeptide of FVIII, as well as also a method for preparing a complex comprising modified FVIII and unmodified VWF by mixing a modified FVIII prepared by the method described above with naturally occurring VWF prepared by the method described above.

The invention also encompasses the use of a modified FVIII such as the one prepared by the method described above and VWF of natural origin for the preparation of a combined pharmaceutical preparation for simultaneous, sequential or separate use in the therapy of bleeding disorders, preferably in the therapy of hemophilia A and / or von Willebrand disease.

The "functional half-life" according to the present invention refers to the half-life of the biological activity of the modified FVIII or of a modified FVIII complex with unmodified VWF once it has been administered to a mammal and can be measured in vitro. in blood samples taken at different time intervals from said mammal after administering the modified FVIII or the modified FVIII complex with unmodified VWF.

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The terms "fuse" or "fused" refer to the addition of amino acids to the C-terminal part of FVIII. When reference is made herein to a "fusion with the C-terminal amino acid of FVIII", this means a fusion with exactly the C-terminal amino acid of FVIII in amino acid 2332 of the naturally occurring mature FVIII cDNA sequence. Mature FVIII refers to the respective polypeptide after excision of the propeptide. However, the invention also encompasses a "fusion with the C-terminal part of FVIII", which within the meaning of this invention may also include respectively a fusion with an FVIII molecule in which one or more amino acid positions have been suppressed until a maximum of n amino acids from the C-terminal amino acid of FVIII. The number n is an integer that should not exceed 5%, preferably should not exceed 1% of the total number of amino acids in FVIII. Normally, n is 20, preferably 15, more preferably 10, even more preferably 5 or less (e.g., 1, 2, 3, 4 or 5).

In one embodiment, the modified FVIII has the following structure:

N -FVIII -C -L1-H, [formula 1]

where

N is an N-terminal part of FVIII,

L1 is a chemical bond or a connecting sequence H is an HLEP and

C is a C-terminal part of FVIII.

L1 may be a chemical bond or a linker sequence consisting of one or more amino acids, e.g. eg, from 1 to 20, from 1 to 15, from 1 to 10, from 1 to 5 or from 1 to 3 (eg, 1, 2 or 3) amino acids, which may be the same or different from each other . Normally, the connecting sequences are not present at the corresponding position of the naturally occurring coagulation factor. Some examples of suitable amino acids present in L1 include Gly and Ser.

The preferred HLEP sequences are described below. Similarly, in the invention, fusions are included with the exact "N-terminal amino acid" of the respective HLEP, or fusions with the "N-terminal part" of the respective HLEP, which includes N-terminal deletions of one or more amino acids of the HLEP.

The modified FVIII or the FVIII complex modified with the unmodified VWF of the invention may comprise more than one HLEP sequence, e.g. eg, two or three sequences of HLEP. These multiple HLEP sequences may be fused with the C-terminal part of FVIII in tandem, e.g. eg, as successive repetitions.

FVIII can be proteolytically processed in several stages. For example, as mentioned above, during its secretion in plasma, the single-chain FVIII is cleaved intracellularly at the B-A3 border and at different sites in domain B. The heavy chain is linked by a metal ion to the light chain that presents the structure of domains A3-C1-C2. FVIII is activated by proteolytic cleavage in amino acids Arg372 and Arg740 of the heavy chain and in Arg1689 of the light chain, which generates the activated FVIII heterotrimer consisting of domain A1, domain A2 and light chain (A3- C1-C2), a 73 kDa fragment. Therefore, the active form of FVIII (FVIIIa) is constituted by an associated A1 subunit through binding to a divalent metal ion to an A3-C1-C2 light chain cleaved by thrombin and a free associated A2 subunit in a manner relatively weak with domain A1 and domain A3.

Accordingly, the present invention further encompasses modified FVIII that is not present as a single stranded polypeptide, but is made up of several polypeptides (e.g., one or two or three) that are associated with each other by non-covalent bonds.

Preferably, N-FVIII-C comprises the full length sequence of FVIII. N-terminal, C-terminal or internal deletions of FVIII are also included, provided that the biological activity of FVIII is preserved. The biological activity is conserved, within the meaning of the invention, if the FVIII with deletions retains at least 10%, preferably at least 25%, more preferably at least 50%, preferably at least 75% of the biological activity of FVIII of natural origin. The biological activity of FVIII can be determined by an expert as described below.

A suitable test to determine the biological activity of FVIII consists, for example, in the one-stage or two-stage coagulation test (Rizza et al. 1982. Coagulation assay of FVIII: C and FIXa in Bloom ed. The Hemophilias. NY Churchchill Livingston 1992) or the FVIII: C chromogenic substrate assay (S. Rosen, 1984. Scand J Haematol 33: 139-145, supl.). The content of these references is incorporated herein by reference.

The cDNA sequence and amino acid sequence of the naturally occurring mature form of the human blood coagulation FVIII are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively. The reference to an amino acid position of a specific sequence refers to the position of said amino acid in the FVIII protein

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of natural origin and does not exclude the presence of mutations, e.g. eg, deletions, insertions and / or substitutions in other positions of the sequence to which reference is made. For example, a mutation in "Glu2004" referring to SEQ ID NO: 15 does not exclude the fact that, in the modified counterpart, one or more amino acids are missing at positions 1-2332 of SEQ ID NO: 15.

The terms "Blood coagulation factor VIII", "Factor VIII" and "FVIM" are used interchangeably herein. The expression "Blood coagulation factor VIII" includes the FVIII of blood clotting of natural origin, as well as derivatives of the FVIII of blood coagulation of natural origin that possess the procoagulant activity of the FVIII of blood coagulation of natural origin The derivatives may contain deletions, insertions and / or additions compared to the naturally occurring amino acid sequence of FVIII. The term FVIII includes proteolytically processed forms of FVIII, e.g. eg, the form before activation, comprising the heavy chain and the light chain.

The term "FVIII" includes any mutants or variants of FVIII containing at least 25%, more preferably at least 50%, and preferably at least 75% of the biological activity of naturally occurring factor VIII.

By way of non-limiting examples, FVIII molecules include FVIII mutants that prevent or reduce APC cleavage (Amano 1998. Thromb. Haemost. 79: 557-563), FVIII mutants that further stabilize the A2 domain (WO 97 / 40145), FVIII mutants that result in increased expression (Swaroop et al. 1997. JBC 272: 24121-24124), FVIII mutants that reduce their immunogenicity (Lollar 1999. Thromb. Haemost. 82: 505- 508), reconstituted FVIII from light and heavy chains expressed differently (Oh et al. 1999. Exp. Mol. Med. 31: 95-100), FVIII mutants that reduce receptor binding that lead to catabolism of FVIII as HSPG (acronym for heparan sulfate proteoglycans) and / or LRP (acronym for a protein related to a low density lipoprotein receptor) (Ananyeva et al. 2001. TCM, 11: 251- 257), FVIII variants stabilized with a disulfide bridge (Gale et al., 2006. J. Thromb. Hemost 4: 1315-1322), FVIII mutants with improved secretion properties (Miao et al., 2004. Blood 103: 3412-3419), FVIII mutants with increased cofactor specific activity (Wakabayashi et al., 2005. Biochemistry 44: 10298-304), FVIII mutants with improved biosynthesis and secretion, reduced interaction with RE chaperone, improved RE-Golgi transport, increased activation or resistance to deactivation and improved half-life (summarized by Pipe 2004. Sem. Thromb. Hemost. 30: 227-237). All these mutants and variants of FVIII are incorporated herein by reference in their entirety.

VWF can be proteolytically processed in several stages. For example, as mentioned above, the ADAMTS13 protease cleaves VWF in the VWF domain A2. Accordingly, the present invention also encompasses a modified VWF that has been proteolytically cleaved, e.g. e.g., by ADAMTS13. Such cleavage would result in multimeric VWF chains that would comprise at least one or at least two VWF monomers at their ends that would have been cleaved by ADAMTS13.

Preferably, N-VWF-C comprises the full length sequence of VWF. N-terminal, C-terminal or internal VWF deletions are also included, provided that the biological activity of VWF is preserved. The biological activity is conserved, within the meaning of the invention, if the VWF with deletions retains at least 10%, preferably at least 25%, more preferably at least 50%, preferably at least 75% of The biological activity of VWF of natural origin. The biological activity of naturally occurring VWF can be determined by an expert using methods to determine the activity of the ristocetin cofactor (Federici AB et al. 2004. Haematology 89: 77-85), the binding of VWF to GP Iba of the glycoprotein complex of platelets Ib-V-IX (Sucker et al. 2006. Clin Appl Thromb Hemost. 12: 305-310) or a collagen binding assay (Kallas and Talpsep. 2001. Annals of Hematology 80: 466-471).

Within the above definition, "FVIII" and / or "VWF" also include natural allelic variations that may exist and may occur from one individual to another. Within the above definition, "FVIII" and / or "VWF" also include variants of FVIII and / or VWF. Such variants differ in one or more amino acid residues of the sequence of natural origin. Examples of such differences may include conservative amino acid substitutions, that is, substitutions within amino acid groups with similar characteristics, e.g. eg, (1) small amino acids, (2) acidic amino acids, (3) polar amino acids, (4) basic amino acids, (5) hydrophobic amino acids and (6) aromatic amino acids. The following table shows some examples of such conservative substitutions.

Table 1:

 (one)

 Alanine Glycine

 (2)

 Aspartic acid Glutamic acid

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 (3)

 Asparagine Serine Glutamine Threonine

 (4)

 Arginine Histidine Lysine

 (5)

 Isoleucine Leucine Methionine Valine

 (6)

 Phenylalanine Tyrosine Tryptophan

One or more HLEP can be fused with the C-terminal part of FVIII preferably, so as not to interfere with the binding capabilities of FVIII, for example, to VWF, platelets or FIX.

Once FVIII has been activated endogenously during coagulation in vivo, it may no longer be desirable to maintain the increased functional half-life of FVIII now activated, as this could lead to thrombotic complications, which is already the case for a coagulation factor. activated from natural origin such as FVIIa (Aledort 2004. J Thromb Haemost 2: 1700-1708) and that could be more relevant if the activated factor had an increased functional half-life. Accordingly, another objective of the present invention is to provide long-lived FVIII molecules, which, after endogenous activation in vivo or after the availability of a cofactor, have a functional half-life comparable to that of unmodified FVIII. This can be achieved, by way of non-limiting example, by introducing a cleavage site, for example, for a coagulation factor between the C-terminal part of FVIII and the HLEP. With such sequences that connect FVIII-HLEP, the activation of the FVIII chimeric protein of the invention will lead to a complete concomitant separation of FVIIIa from the rest of HLEP. Accordingly, in one embodiment, the functional half-life of endogenously activated modified FVIII is substantially the same as that of activated natural origin FVIII (eg, ± 15%, preferably ± 10%).

In yet another embodiment of the invention, however, one or more of the proteolytic cleavage sites, preferably the thrombin cleavage sites in Arg 740 and / or Arg372, are mutated or suppressed in order to avoid cleavage and this gives as a result an insertion protein that has improved properties as an improved functional half-life even as an activated molecule.

In another embodiment of the invention, the FVIII proteins of the invention can be expressed as two separate chains (see below).

The modified FVIII according to this invention may be a single stranded polypeptide, or it may consist of two or three polypeptide chains that are associated by non-covalent bonds, due to proteolytic processing.

In another embodiment of the invention, amino acids from or near the PACE / Furina cleavage site (Arg1648) are mutated or suppressed in order to avoid cleavage by PACE / Furina. It is believed that this results in a single-chain FVIII / HLEP fusion molecule with an improved half-life.

In one embodiment of the invention, the modified FVIII of the invention exhibits a greater functional half-life compared to the corresponding FVIII form that does not contain integrated HLEP and / or the naturally occurring FVIII form. The functional half-life can be determined, e.g. eg, in vivo in animal models of hemophilia A, such as mice in which FVIII has been suppressed, in which a longer hemostatic effect could be expected compared to naturally occurring FVIII. The hemostatic effect could be evaluated, for example, by determining the time needed to stop the bleeding after the tail clamp.

The functional half-life in one embodiment of the invention is the half-life of the biological activity of FVIII once it has been administered to a mammal and measured in vitro. The functional half-life of the modified FVIII according to the invention is higher than that of the FVIII that lacks the modification, as evaluated in the same species. The functional half-life preferably increases at least 10%, preferably 25%, more preferably at least 50% and even more preferably at least 100% compared to the natural origin form of FVIII.

The functional half-life of a modified FVIII comprising a modification of HLEP can be determined by administering the respective modified FVIII (and in comparison the FVIII of natural origin) to rats, rabbits or other experimental animal species intravenously or subcutaneously and following elimination of the biological activity of said modified or, unmodified, coagulation factor in blood samples taken at appropriate intervals after application. Some suitable test methods are the activity tests described herein.

As an alternative marker for the half-life of biological activity, the levels of modified FVIII antigen or, respectively, of natural origin, or levels of naturally occurring VWF can also be measured. For the

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therefore, the invention also includes modified FVIII molecules that contain a fusion with an HLEP in the C-terminal part of FVIII, characterized in that the modified FVIII or the modified FVIII complex with unmodified VWF has a prolonged half-life of the antigen of FVIII compared to the half-life of the FVIII antigen that lacks such insertion. The "half-life of the FVIII antigen" according to the present invention is the half-life of the FVIII antigen once it has been administered to a mammal and measured in vitro. The expert will know antigen test methods based on specific antibodies in an enzyme immunoassay format and these can be purchased from commercial suppliers (eg, Dade Behring, Instrumentation Laboratory, Abbott Laboratories, Diagnostic Stago). The functional and antigen half-lives can be calculated using the evaluation points of the beta phase of elimination according to the formula t-i / 2 = In2 / k, where k is the slope of the regression line.

In another embodiment, the functional half-life of the endogenously activated modified FVIII is prolonged compared to that of the naturally occurring activated FVIII. The increase may be greater than 15%, for example, at least 20% or at least 50%. Again, such functional half-life values can be measured and calculated as described for the functional half-lives above. Increased half-lives of endogenously activated modified FVIII molecules may be beneficial in situations where only very low levels of FVIII are available that, therefore, are not thrombogenic. Such situations may occur, e.g. eg, after a gene therapy treatment, in which often only low expression rates can be obtained. Therefore, such stabilized FVIII molecules could be beneficial, e.g. eg, in gene therapy, despite the thrombogenic risk associated with such FVIII molecules if they are administered as proteins in physiological or high doses.

In another embodiment of the invention, the modified FVIII of the invention exhibits an improved in vivo recovery compared to the naturally occurring FVIII. In vivo recovery can be determined in vivo, for example, in normal animals or in animal models of hemophilia A, such as mice in which FVIII has been suppressed, in which it would be expected to detect an increase in the percentage of modified FVIII of the invention by activity tests or circulating antigen shortly after (5 to 10 min) after iv administration in comparison with the corresponding FVIII of natural origin or the VWF of natural origin.

In vivo recovery preferably increases at least 10%, more preferably at least 20% and even more preferably at least 40% compared to the naturally occurring FVIII form or with naturally occurring VWF.

In yet another embodiment of the invention, constant regions of immunoglobulin or their portions are used as HLEP. Preferably, the Fc region comprising a CH2 and CH3 domain and a hinge region of an IgG is used, more preferably of an IgG1 or its fragments or variants, including variant variants that improve neonatal Fc receptor binding (FcRn).

Another objective of the present invention is to provide long-lived FVIII molecules that, after in vivo proteolytic processing, have a functional half-life comparable to that of an unmodified FVIII. This can be achieved by maintaining certain cleavage sites in the modified FVIII that cause proteolytic cleavage, for example, when they come into contact with activated coagulation factors, which separate the FVIII from the HLEP. Accordingly, in one embodiment, the functional half-life of the proteolytically processed modified FVIII is substantially the same as that of the unmodified VWF that lacks the modification and / or is substantially the same as that of the naturally occurring VWF (e.g., ± 15%, preferably ± 10%).

A further embodiment of the invention are modified FVIII polypeptides that are fused with an HLEP, for example, albumin in the C-terminal part of the FVIII molecule, which exhibit a reduction in VWF binding or do not bind to VWF in absolute.

Connecting sequences

In accordance with this invention, the therapeutic polypeptide moiety can be coupled with the rest of HLEP via a peptide linker. The connector must be non-immunogenic and can be a cleavable or non-cleavable connector.

Non-cleavable connectors may comprise alternate glycine and serine residues as illustrated in WO2007 / 090584.

In another embodiment of the invention, the peptide linker between the rest of FVIII and / or VWF and the rest of albumin is constituted by peptide sequences, which serve as connectors between natural domains in human proteins. Preferably, such peptide sequences in their natural environment are located near the surface of the protein and can be accessed by the immune system, so that a natural tolerance against this sequence can be assumed. Examples are provided in WO2007 / 090584.

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The cleavable connectors must be flexible enough to allow cleavage by proteases. In a preferred embodiment, the cleavage of the linker proceeds in a comparatively rapid manner as the activation of FVIII in the fusion protein, if the fusion protein is a modified FVIII.

The cleavable connector preferably comprises a sequence derived from

a) the therapeutic polypeptide to be administered per se, if it contains proteolytic cleavage sites that are proteolytically cleaved during activation of the therapeutic polypeptide,

b) a substrate polypeptide cleaved by a protease that is activated or formed by the intervention of the therapeutic polypeptide,

c) a polypeptide that is involved in coagulation or fibrinolysis.

The connecting region, in a more preferred embodiment, comprises a sequence of FVIII and / or VWF, which should result in a reduction of the risk of obtaining neoantigenic properties of the expressed fusion protein. In addition, in the case that the therapeutic protein is FVIII, which needs to be proteolytically activated, the kinetics of cleavage of the peptide linker will more closely reflect the activation kinetics related to the coagulation of the zymogen.

In a preferred embodiment, the therapeutic polypeptide is an FVIII zymogen and the HLEP is albumin. In this case, the connecting sequence is either derived from the sequences of the activated regions of FVIII, from the cleavage region of any FIX substrate such as FX or FVIII, or from the cleavage region of any substrate polypeptide that is cleaved by a protease in whose activation FIXa intervenes.

In a very preferred embodiment, the linker peptide is derived from FVIII itself and comprises sequences that encompass thrombin cleavage sites at amino acid positions 372, 740 and 1689 of SEQ ID NO. 15, respectively. In another preferred embodiment, the linker peptide is derived from FX, FIX, FVII or FXI.

The connecting peptides are preferentially cleaved by the coagulation system proteases, for example, Flla, FIXa, FXa, FXla, FXlla and FVlla.

Such connecting sequences can also be used in the modified VWF of the invention.

Some illustrative combinations of the therapeutic polypeptide, cleavable linker and HLEP include the constructs listed in WO2007 / 090584 (for example, in Table 2 and Figure 4) and WO2007 / 144173 (for example, in Table 3a and 3b ), but are not limited to these.

Polypeptides that improve half-life (HLEP)

A "half-life enhancing polypeptide", as used herein, is selected from the group consisting of albumin. It may be a protein that improves the full-length half-life described herein or one or more of its fragments that are capable of stabilizing or prolonging the therapeutic activity or the biological activity of the clotting factor. Such fragments may be 10 or more amino acids in length or may include at least about 15, at least about 20, at least about 25, at least about 30, at least about 50, at least about 100 or more contiguous amino acids of the sequence of HLEP or may include all or part of the specific domains of the respective HLEP, provided that the HLEP fragment provides an extension of the functional half-life of at least 25% compared to a naturally occurring FVIII or naturally occurring VWF.

The HLEP portion of the proposed coagulation factor insertion constructs of the invention may be a variant of a normal HLEP. The term "variants" includes insertions, deletions and substitutions, whether conservative or non-conservative, where such changes do not substantially alter the active center or active domain that confers the biological activities of the modified FVIII or modified VWF.

In particular, the proposed FVIII HLEP or VWF HLEP fusion constructs of the invention may include naturally occurring polymorphic variants of HLEP and HLEP fragments. HLEP can be derived from any vertebrate, especially any mammal, for example, a human being, monkey, cow, sheep or pig. Non-mammalian HLEPs include, but are not limited to, those of chicken or salmon.

Albumin as HLEP

The terms "human serum albumin" (ASH), "human albumin" (AH) and "albumin" (ALB) are used interchangeably in this application. The terms "albumin" and "serum albumin" are broader and encompass human serum albumin (and its fragments and variants), as well as albumin of other species (and their fragments and variants).

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The term "albumin," as used herein, collectively refers to an amino acid or polypeptide sequence of albumin, or albumin fragment or variant, with one or more functional activities (eg, biological activities ) of albumin. In particular, the term "albumin" refers to human albumin or its fragments, especially the mature form of human albumin as shown in SEQ ID NO: 16 herein, or albumin of other vertebrates or their fragments, or the like. or variants of these molecules or their fragments.

In particular, the proposed FVIII fusion constructs of the invention may include naturally occurring polymorphic variants of human albumin and human albumin fragments. In general terms, a fragment or variant of albumin will be at least 10 in length, preferably at least 40 and more preferably more than 70 amino acids. The albumin variant may preferably be constituted or may alternatively comprise at least one entire albumin domain or fragments of said domains, for example, domains 1 (amino acids 1-194 of SEQ ID NO: 16), 2 (amino acids 195-387 of SEQ ID No: 16), 3 (amino acids 388-585 of SEQ ID NO: 16), 1 + 2 (1-387 of SEQ ID NO: 16), 2 + 3 (195-585 of SEQ ID NO: 16) or 1 + 3 (amino acids 1-194 of SEQ ID NO: 16 + amino acids 388-585 of SEQ ID NO: 16). Each domain is constituted in turn by two homologous subdomains, namely 1-105, 120-194, 195-291, 316-387, 388-491 and 512-585, with connecting regions between flexible domains comprising Lys106 residues to Glu119, Glu292 to Val315 and Glu492 to Ala511.

The albumin portion of the proposed FVIII fusion constructs of the invention may comprise at least one subdomain or domain of AH or conservative modifications thereof.

Polynucleotides

The invention further relates to a polynucleotide encoding a modified coagulation factor, preferably a modified FVIII variant as described in this application. The term "polynucleotide (s)" generally refers to any polyiribonucleotide or polydeoxyribonucleotide that can be RNA or unmodified DNA or modified RNA or DNA. The polynucleotide can be single- or double-stranded DNA, or single- or double-stranded RNA. The term "polynucleotide (s)," as used herein, also includes DNA or RNA that comprise one or more modified bases and / or unusual bases such as inosine. It will be appreciated that various modifications to DNA and RNA can be made with many useful purposes known to those skilled in the art. The term "polynucleotide (s)", as used herein, encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example. , simple and complex cells.

The expert will understand that, due to the degeneracy of the genetic code, a given polypeptide can be encoded by different polynucleotides. These "variants" are encompassed in this invention.

Preferably, the polynucleotide of the invention is an isolated polynucleotide. The term "isolated" polynucleotide refers to a polynucleotide that is substantially free of other nucleic acid sequences such as, without limitation, other chromosomal and extrachromosomal DNA and RNA. Isolated polynucleotides can be purified from a host cell. Conventional nucleic acid purification methods known to those skilled in the art can be used to obtain the isolated polynucleotides. The term also includes recombinant polynucleotides and chemically synthesized polynucleotides.

The invention further relates to a group of polynucleotides that jointly encode the modified FVIII and / or the modified VWF of the invention. A first polynucleotide of the group can encode the N-terminal part of the modified FVIII and a second polynucleotide can encode the C-terminal part of the modified FVIII.

A further aspect of the invention consists of a plasmid or vector comprising a polynucleotide according to the invention. Preferably, the plasmid or vector is an expression vector. In a particular embodiment, the vector is a transfer vector for use in human gene therapy.

The invention also relates to a group of plasmids or vectors comprising the above group of polynucleotides. A first plasmid or vector may contain said first polynucleotide and a second plasmid or vector may contain said second polynucleotide. By way of example and with reference to the FVIII coagulation factor, the signal peptide coding sequences, domains A1 and A2, the rest of the B domain sequence and the HLEP can be cloned into the first expression vector and the coding sequences of A3, C1 and C2 with a suitable signal peptide sequence can be cloned into the second expression vector. Both expression vectors are co-transfected in a suitable host cell, which will lead to the expression of the light and heavy chains of the FVIII molecule of the invention and the formation of a functional protein.

Alternatively, the FVIII signal peptide coding sequence, the A1 and A2 domains are cloned into the first expression vector and the FLEII HLEP, A3, C1 and C2 coding sequences with a suitable signal peptide sequence are cloned into the Second expression vector. Both expression vectors are co-transfected in a

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Suitable host cell, which will lead to the expression of the light and heavy chains of the FVIII molecule of the invention and the formation of a functional protein.

Alternatively, both coding sequences are cloned into an expression vector, either using two separate promoter sequences or a promoter and an internal ribosome entry site element (IRES) to direct the expression of both FVIII chains.

A further aspect of the invention consists of a host cell comprising a polynucleotide, a plasmid or vector of the invention or a group of polynucleotides or a group of plasmids or vectors as described herein.

The host cells of the invention can be used in a method to produce a modified coagulation factor, preferably a modified FVIII molecule, which is part of this invention. The method comprises:

(a) culturing host cells of the invention under conditions such that the desired insertion protein is expressed; Y

(b) optionally recovering the desired insertion protein from the host cells or from the culture medium.

It is preferred to purify the modified FVIII of the present invention to obtain a purity> 80%, more preferably a purity> 95% and a pharmaceutically pure state that is greater than 99.9% purity with respect to contaminating macromolecules, in particular, is particularly preferred. other proteins and nucleic acids, and free of infectious agents and pyrogens. Preferably, an isolated or purified modified FVIII of the invention is substantially free of other unrelated polypeptides.

The different products of the invention are useful as medicaments. Accordingly, the invention relates to a pharmaceutical composition comprising a modified FVIII as described herein, a polynucleotide of the invention or a plasmid or vector of the invention.

The invention also relates to a method for treating an individual suffering from a blood clotting disorder such as hemophilia A or B. The method comprises administering to said individual an effective amount of FVIII or VWF-modified FVIII complex. not modified as described herein. In another embodiment, the method comprises administering to the individual an effective amount of a polynucleotide of the invention or of a plasmid or vector of the invention. Alternatively, the method may comprise administering to the individual an effective amount of the host cells of the invention described herein.

Expression of the proposed mutants

The production of recombinant mutated proteins at high levels in suitable host cells requires the coupling of the aforementioned modified cDNAs into effective transcription units together with suitable regulatory elements in a recombinant expression vector that can be propagated in various expression systems according to methods known to those skilled in the art. Effective transcriptional regulatory elements could be derived from viruses that have animal cells as their natural hosts or from the chromosomal DNA of animal cells. Preferably, promoter-enhancer combinations derived from simian 40 virus, adenovirus, BK polyoma virus, human cytomegalovirus or long terminal repeat of Rous sarcoma virus, or promoter-enhancer combinations that include form-transcribed genes can be used strongly constitutive in animal cells such as beta-actin or GRP78. In order to achieve stable high levels of mRNA transcribed from the cDNAs, the transcription unit must contain in its 3'-proximal part a region of DNA encoding a polyadenylation-termination sequence of transcription. Preferably, this sequence is derived from the early transcription region of simian virus 40, the rabbit beta-globin gene or the human tissue plasminogen activator gene.

Next, the cDNAs are integrated into the genome of a suitable host cell line for the expression of the modified FVIII and / or VWF proteins. Preferably, this cell line should be an animal cell line of vertebrate origin in order to ensure correct folding, the formation of disulfide bridges, glycosylation related to asparagine and other post-translational modifications, as well as secretion in the culture medium. Some examples of other post-translational modifications are tyrosine O-sulphation and proteolytic processing of the nascent polypeptide chain. Some examples of cell lines that can be used are primate COS cells, mouse L cells, mouse C127 cells, hamster BHK-21 cells, human embryonic kidney 293 cells and hamster CHO cells.

The recombinant expression vector encoding the corresponding cDNAs can be introduced into an animal cell line in several different ways. For example, recombinant expression vectors can be created from vectors based on different animal viruses. Some examples of these are vectors based on baculovirus, vaccinia virus, adenovirus, and preferably bovine papillomavirus.

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Transcription units encoding the corresponding DNAs can also be introduced into animal cells together with another recombinant gene that can act as a dominant selectable marker in these cells in order to facilitate the isolation of specific cell clones that have integrated the recombinant DNA into their genome Some examples of this type of dominant selectable marker genes are Tn5 aminoglycoside phosphotransferase, which confers resistance to geneticin (G418), hygromycin phosphotransferase, which confers resistance to hygromycin, and puromycin acetyltransferase, which confers resistance to Puromycin The recombinant expression vector encoding such a selectable marker may reside in either the same vector, as a cDNA encoder of the desired protein, or it may be encoded by a different vector that is simultaneously introduced and integrated into the genome of the host cell, which often results in a close physical connection between the different transcription units.

Other types of selectable marker genes that can be used in conjunction with the cDNAs of the desired protein are based on several transcription units encoding dihydrofolate reductase (dhfr). After the introduction of this type of gene into cells that lack endogenous dhfr activity, preferably CHO cells (DUKX-B11, DG-44), this will allow said cells to grow in nucleoside-free media. An example of such a medium is Ham F12 without hypoxanthine, thymidine or glycine. These dhfr genes can be introduced together with the FVIII cDNA transcription units in CHO cells of the above type, either linked to the same vector or in different vectors, thereby creating dhfr-positive cell lines that produce recombinant protein.

If the above cell lines are cultured in the presence of the cytotoxic dhfr inhibitor methotrexate, new methotrexate resistant cell lines will emerge. These cell lines can produce recombinant protein with a higher rate, due to the amplified number of dhfr that has been bound and the transcription units of the desired protein. When these cell lines are propagated in increasing concentrations of methotrexate (1-10000 nM), new cell lines can be obtained that produce the desired protein at a very high rate.

The above cell lines that produce the desired protein can be grown on a large scale, either in a suspension culture or on several solid supports. Some examples of these supports are microcarriers based on dextran or collagen matrices, or solid supports in the form of hollow fibers or various ceramic materials. When grown in a suspension cell culture or in microcarriers, the culture of the above cell lines can be carried out either as a culture in a bath or as a perfusion culture with the continuous production of conditioned medium during periods of prolonged time Therefore, in accordance with the present invention, the above cell lines are suitable for the development of an industrial process for the production of the desired recombinant mutated proteins.

Purification and formulation

The recombinant modified FVIII, which accumulates in the medium of secretory cells of the above types, can be concentrated and purified by various biochemical and chromatographic methods, which include methods that employ differences in size, charge, hydrophobicity, solubility, specific affinity, etc. between the desired protein and other substances in the cell culture medium.

An example of such purification is the adsorption of the recombinant mutated protein in a directed monoclonal antibody, e.g. eg, to a human albumin HLEP, or directed to the respective coagulation factor, which is immobilized on a solid support. After adsorption of the modified FVIII on the support, its washing and desorption, the protein can be further purified by various chromatographic techniques based on the above properties. The order of the purification steps is selected, e.g. eg, according to the capacity and selectivity of the steps, the stability of the support or other aspects. Some preferred purification steps, e.g. For example, they are, without limitation, ion exchange chromatography steps, immune affinity chromatography steps, affinity chromatography steps, hydrophobic interaction chromatography steps, dye chromatography steps, hydroxyapatite chromatography steps, multimodal chromatography and size exclusion chromatography steps.

In order to minimize the theoretical risk of contamination with viruses, additional steps can be included in the process that allow the effective deactivation or elimination of viruses. Such steps are, e.g. eg, a heat treatment in a liquid or solid state, a treatment with solvents and / or detergents, radiation in the visible or UV spectrum, gamma radiation or nanofiltration.

The modified polynucleotides (eg, DNA) of this invention can also be integrated into a transfer vector for use in human gene therapy.

The different embodiments described herein can be combined with each other. The present invention will be further described in more detail in the examples thereof shown below. This description of specific embodiments of the invention will be carried out in conjunction with the attached figures.

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The modified FVIII described in this invention can be formulated in pharmaceutical preparations for therapeutic use. The purified protein can be dissolved in conventional physiologically compatible aqueous buffer solutions, to which, optionally, pharmaceutical excipients can be added to provide pharmaceutical preparations.

Such pharmaceutical carriers and excipients, as well as suitable pharmaceutical formulations, are in common use in the art (see, for example, "Pharmaceutical Formulation Development of Peptides and Proteins", Frokjaer et al., Taylor and Francis (2000) or "Handbook of Pharmaceutical Excipients", 3rd edition, Kibbe et al., Pharmaceutical Press (2000)). In particular, the pharmaceutical composition comprising the polypeptide variant of the invention can be formulated in a stable or lyophilized liquid form. The polypeptide variant can be lyophilized by several methods known in the art. Lyophilized formulations are reconstituted before use by the addition of one or more pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline.

Composition formulations are supplied to the individual by any pharmaceutically suitable method of administration. Several delivery systems are known and can be used to administer the composition by any convenient route. Preferably, the compositions of the invention are administered systemically. For systemic use, the insertion proteins of the invention are formulated for parenteral delivery (eg, intravenous, subcutaneous, intramuscular, intraperitoneal, intracerebral, intrapulmonary, intranasal or transdermal) or enteric (eg oral, vaginal or rectal) according to conventional methods. The most preferred routes of administration are intravenous and subcutaneous administration. The formulations can be administered continuously by infusion or by bolus injection. Some formulations encompass slow release systems.

The insertion proteins of the present invention are administered to patients with a therapeutically effective dose, which refers to a dose that is sufficient to produce the desired effects, so as to prevent or reduce the severity or spread of the condition or indication. that is being treated without reaching a dose that produces intolerable adverse side effects. The exact dose depends on many factors such as, e.g. eg, the indication, formulation, route of administration, and should be determined in clinical and preclinical trials for each respective indication.

The pharmaceutical composition of the invention can be administered alone or in combination with other therapeutic agents. These agents can be incorporated as part of the same pharmaceutical preparation. An example of such an agent is the combination of modified FVIII with unmodified VWF.

Figures

Figure 1: Antigen levels and activity of naturally occurring FVIII and FVIII-C-terminal-albumin fusion polypeptides.

Figure 2: Comparison of the pharmacokinetic parameters of human FVIII: Ag in mice with VWF suppression after i.v. 100 U (FVIII: Ag) / kg of FVIII of natural origin and FVIII-FP 1656 VWF (mean; n = 4 / evaluation point).

Figure 3: Proportions of VWF: RCo / VWF: Ag of cell culture supernatants containing naturally occurring rVWF (1570/1212), rVWF-FP (1572/1212) containing albumin bound to the C-terminal end, or a culture mixed expression cell containing a mixture of naturally occurring rVWF (1570/1212) and rVWF-FP (1572/1212) transfected with a 5: 1 ratio. Values of approximately 0.8 were obtained in each case, which are close to 1, which corresponds to the theoretical proportion of NHP according to the definitions of the units.

Figure 4: Electrophoresis in agarose-SDS gel of naturally occurring rVWF (1570/1212) expressed in HEK (B) and rVWF-FP (1572/1212) cells expressed in HEK (A) cells. Bands were detected using either VWF or albumin (ASH) antibodies.

Figure 5: Comparison of the pharmacokinetic parameters of human rWVF of natural origin and rVWF-FP after i.v. of 100 IU of VWF: Ag in rats (mean, n = 2-3 / evaluation point).

Examples:

Example 1: Generation of expression vectors for FVIII molecules with C-terminal albumin fusion

First, a pIRESpuro3-based expression plasmid (BD Biosciences) containing the full-length FVIII cDNA sequence at its multiple cloning site (pF8-FL) was used to create an FVIII with the deleted B domain. To this end, oligonucleotides F8-1 and F8-2 (SEQ ID NO 1 and 2) were used in a site-directed mutagenesis experiment according to standard protocols (site-directed mutagenesis kit QuickChange XL, Stratagene, La Jolla , CA, USA) using pF8-FL as a template to eliminate domain B.

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A second step, a sequence was introduced that encoded the RRGR amino acid sequence to connect R740 of domain A2 with R1648 of domain a3. This was carried out in another round of site-directed mutagenesis using primers F8-3 and F8-4 (SEQ ID NO 3 and 4). The resulting plasmid was named pF8-457. A stepwise FVIII-albumin fusion construct was generated. First, a PinAl cleavage site was introduced at the 3'-terminal end of FVIII. To this end, a PCR fragment was generated using pF8-457 as a template, using the PCR primers We2827 and We2828 (SEQ ID NO 5 and 6), which was subsequently gel purified, cut with the restriction endonucleases BspE1 and Notl, and bound in pF8-457 previously digested with BspE1 and Notl. The resulting plasmid (pF8-1433) was then cut with the PinAl and Notl enzymes, and a fragment obtained by pCr was inserted into a plasmid containing human albumin cDNA using primers We 2829 and We 2830 (SEQ ID NO 7 and 8), and subsequently digested with the enzymes PinAl and Notl. The resulting expression plasmid (pF8-1434) contained the coding sequences for an FVIII with the deleted B domain followed by a PinAl site to insert connectors (encoding the ThrGly amino acid sequence) and the human albumin coding sequence. The amino acid sequence encoded by pF8-1434 is represented as SEQ ID NO 9.

Next, the connecting sequences that separate the rest of FVIII and the rest of albumin could easily be inserted into the newly created PinAI site described above. The following describes the insertion of two connecting sequences. In addition, based on pF8-1434, the TG connector could be completely removed and even deletions at the C-terminal end of FVIII or the N-terminal end of albumin can be carried out using site-directed mutagenesis.

Insertion of a cleavable linker derived from the thrombin cleavage site of FVIII: first, a PCR fragment was generated containing the sequence encoding the thrombin cleavage site at position 372 by PCR using primers We2979 and We2980 (SEQ ID NO 10 and 11) and pF8-457 as a mold. This fragment was purified, digested with PinAl and ligated into pF8-1434 digested with PinAl. Sequencing verified the insertion of the correct orientation of the fragment and the resulting plasmid was designated pF8-1563.

Insertion of a flexible glycine / serine connector: a PCR fragment containing the coding sequence for a 31 amino acid glycine / serine connector was amplified by PCR from pFVII-937 described in WO2007 / 090584 using We2991 primers and We2992 (SEQ ID No. 12 and 13). This fragment was then purified, digested with the restriction endonuclease PinAl and ligated into pF8-1434 digested with PinAl. Sequencing verified the insertion of the correct orientation of the fragment and the resulting plasmid was designated pF8-1568.

Using the protocols and plasmids described above and applying molecular biology techniques known to those skilled in the art (and described, e.g., in Current Protocols in Molecular Biology, Ausubel FM et al. (Eds.) John Wiley & Sons, Inc .;
http://www.currentprotocols.com/WileyCDA/), an expert can prepare other constructs to replace the albumin with another HLEP or insert any other connector in the described PinAl site. Transfer of the FVIII / albumin cDNA into suitable vectors such as pIRESneo3 (Invitrogen) and pEE12.4 (Lonza) allowed the expression and selection of clones expressing the respective FVIII-albumin fusion protein in CHO cells.

Example 2: Transfection and expression of FVIII and VWF proteins

Expression plasmids were grown in E. coli TOP10 (Invitrogen, Carlsbad, CA, USA) and purified using standard protocols (Qiagen, Hilden, Germany). HEK-293 cells (Invitrogen) were transfected using the Lipofectamine 2000 reagent (Invitrogen) and grown in serum free medium (Invitrogen 293 Express) in the presence of 4 pg / mL of Puromycin and optionally 0.5 IU / mL of VWF. CHO cells (CHO-S, Invitrogen; CHOK1SV, Lonza) were transfected using Lipofectamine 2000 reagent (Invitrogen) and cultured in serum-free medium (Invitrogen CD CHO, 6 mM glutamine for ChO-S and CD-CHO for CHoK1sV) in the presence of 500-1000 pg / mL of Geneticin (CHO-S only). For the expression of FVIII, 0.5 IU / mL of VWF was optionally added. For vWF expression, an expression plasmid encoding PACE / furin (pFu-797) was co-transfected as described in WO2007 / 144173. In another experiment, two plasmids encoding VWF of natural origin and VWF fused at the C-terminal end with albumin were co-transfected with pFu-797 to obtain VWF multimers with VWF monomers of natural origin and VWF monomers fused with albumin (refer to figure 3). The transfected cell populations were spread by means of cell culture bottles in rotary bottles or small-scale fermenters, from which supernatants were collected for purification.

Table 2 lists the HEK-293 expression data for the constructs described in example 1.

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Table 2:

 Construct

 Activity [U / mL]

 pF8-457

 1.54

 pF8-457 + 0.5 U / mL of VWF

 1.66

 pF8-1434

 1.59

 pF8-1434 + 0.5 U / mL of VWF

 1.82

 pF8-1563 + 0.5 U / mL of VWF

 2.04

 pF8-1568 + 0.5 U / mL of VWF

 1.21

Example 3: Increase in the expression rate of the FVIM-albumin fusion protein

Figure 1 summarizes the results of an expression study of an FVIN-albumin fusion protein in a serum-free cell culture. HEK-293 cells were transfected in triplicate with pF8-1434 (FVIII-C-terminal-albumin fusion) and pF8-457 (naturally occurring FVIII), respectively, seeded in T80 bottles with equivalent cell numbers and grown in the absence of vWf stabilizer. Next, the culture supernatant was collected after 96, 120 and 144 hours, and evaluated to determine FVIII activity.

The results showed an effect enhancing the expression of the albumin residue when it is present as an integral part of the FVIII molecule in cell culture. As a consequence, productivity was clearly improved in the case of the fusion protein compared to the naturally occurring FVIII (Figure 1).

Example 4: Purification of FVIII proteins

A sufficient amount of an immune affinity resin was added to the expression supernatant containing the FVIII molecule to bind FVIII activity almost completely. The immune affinity resin was prepared by covalently attaching a suitable anti-FVIII MAb to a Sephacryl S1000 resin used as a support. After washing the resin, it was placed on a chromatographic column and washed again. Elution was carried out using a buffer containing 250 mM CaCl2 and 50% ethylene glycol.

Immunity affinity chromatography (IAC) fractions containing FVIII: C activity were pooled, subjected to dialysis against a formulation buffer (excipients: sodium chloride, sucrose, histidine, calcium chloride and Tween 80) and concentrated. The samples were either stored frozen or lyophilized using a suitable lyophilization cycle.

Alternatively, the cell culture supernatant containing FVIII is concentrated / purified by ion exchange chromatography first and then by further purification using immune affinity chromatography (IAC). In this case, the eluted material of the ion exchange chromatography is introduced into an IAC column using the resin mentioned above.

Example 5: FVIII activity and antigen analysis

To determine the activity of FVIII: C in vitro, either a coagulation assay (e.g., FVIII-deficient plasma and Pathromtin SL reagent supplied by Dade Behring, Germany) or a chromogenic assay (e.g. ., Coamatic FVIII: C test supplied by Haemochrom). The tests were carried out in accordance with the manufacturer's instructions.

The FVIII antigen (FVIII: Ag) was determined by ELISA, whose performance is known to those skilled in the art. In summary, microplates were incubated with 100 pL per well of the capture antibody (human sheep anti-FVIII IgG, Cedarlane CL20035K-C, with a 1: 200 dilution factor in Buffer A [Sigma C3041]) for 2 hours at room temperature . After washing the plates three times with buffer B (Sigma P3563), serial dilutions of the test sample were incubated in diluent buffer for the sample (Cedarlane), as well as serial dilutions of an FVIII preparation (CSL Behring; 200-2 mU / mL) in diluent buffer for the sample (volumes per well: 100 pL), for two hours at room temperature. After three washing steps with buffer B, 100 pL of a 1: 2 dilution in buffer B of the detection antibody (human anti-FVIII IgG) was added

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of sheep, Cedarlane CL20035K-D, labeled with peroxidase) to each well and incubated for another hour at room temperature. After three washing steps with buffer B, 100 pL of substrate solution (1:10 (v / v) TMB OUVF: TMB OUVG buffer, Dade Behring) was added per well and incubated for 30 minutes at room temperature in Darkness. The addition of 100 pL of stop solution (Dade Behring, OSFA) prepared the samples for reading in a suitable microplate reader at a wavelength of 450 nm. Next, the concentrations of the test samples were calculated using the standard curve with the FVIII preparation as a reference.

Example 6: Evaluation of the pharmacokinetic parameters of FVIII-FP in mice in which VWF has been suppressed after a single i.v.

In order to compare the pharmacokinetic parameters of naturally occurring FVIII (DNA 457) and C-terminal FVIII-FP (DNA 1656), both FVIII variants were administered intravenously to mice. A mouse strain was selected in which VWF had been suppressed (Denis C. et al., Proc. Natl. Acad. Sci. USA, 1998, Vol. 95, 9524-9529) since, among other functions, VWF acts as a carrier and stabilizing protein for FVIII, it thus protects FVIII against its premature degradation, e.g. eg, by proteases, and against their premature elimination of circulation. For an unmodified FVIII, an undisturbed interaction with VWF is essential, as illustrated by cases of hemophilia A, caused by a mutation in the C-terminal region, which causes a reduction in VWF binding. In the case of modified FVIII, such a union may, however, even be unwanted, in order to examine or achieve improved pharmacokinetic parameters. Therefore, both products were injected via i.v. with a dose of 100 U (FVIII: Ag) / kg bolus to two groups of mice (table 3). Blood samples were taken retroorbitally at appropriate intervals, starting 5 minutes after the application of the test substances and up to 24 hours. A blood / mouse sample was taken, processed until plasma was obtained and stored frozen at -20 ° C until analysis. The concentration of human FVIII: Ag was quantified using an ELISA assay specific for human FVIII or by a mixed ELISA specific for FVIII and human albumin, respectively. The mean plasma concentration of the pooled samples for each evaluation point was used to calculate the pharmacokinetic parameters. The half-life was calculated using the evaluation points of the beta phase of the elimination according to the formula t-i / 2 = In2 / k, where k is the slope of the regression line. The result is represented in Figure 2. Surprisingly, FVIII-FP 1656 (ti / 2 = 3.06 h, between 5 and 960 min) presented a terminal half-life approximately 3-4 times longer compared to FVIII of natural origin (ti / 2 = 0.8 h, between 5 and 240 min). In addition, the recovery of FVIII-FP 1656 was increased by approximately 20% compared to FVIII of natural origin (Table 4).

Table 3: Treatment groups for the comparison of FVIII pharmacokinetic parameters in mice in which VWF has been suppressed

 Treatment

 Dose (FVIIkC) / volume / program / via N

 FVIII of natural origin

 100 U (FVIII: Ag) / kg / 0.2 mL / 20 g body weight / t = 0 h / i.v. 24

 FVIII-FP 1656

 100 U (FVIII: Ag) / kg / 0.2 mL / 20 g body weight / t = 0 h / i.v. 24

Table 4: Bioavailability (%) of FVIII of natural origin and modified FVIII, FVIII-FP 1656,

after injection i.v. in mice in which VWF has been suppressed

 Treatment

 Bioavailability (%)

 FVIII of natural origin

 100

 FVIII-FP 1656

 120.4

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Example 7: Generation of expression vectors for naturally occurring VWF and VWF-albumin fusion proteins

First, an expression plasmid was generated that contained the full-length VWF cDNA sequence at its multiple cloning site. To this end, the VWF coding sequence was amplified by polymerase chain reaction (PCR) using the VWF + and VWF- primer set (SEQ ID NO. 17 and 18) under standard conditions known to those skilled in the art (and as described, eg, in Current Protocols in Molecular Biology, Ausubel FM et al. (eds.) John Wiley & Sons, Inc .;
http://www.currentprotocols.com/WileyCDA/) from a plasmid containing VWF cDNA (which can be obtained from commercial suppliers, e.g., pMT2-VWF from ATCC, No. 67122). The resulting PCR fragment was digested with the EcoRI restriction endonuclease and ligated into the pIRESpuro3 expression vector (BD Biosciences, Franklin Lakes, NJ, USA) that had been linearized with EcoRI. The resulting expression plasmid containing the naturally occurring cDNA of VWF after the CMV promoter was named pVWF-1570.

A PCR fragment containing the coding sequence for a 31 amino acid glycine / serine linker and the human albumin cDNA was amplified from pFVII-937 described in WO2007 / 090584 using primers We2994 and We1335 (SEQ ID NO .19 and 20). This PCR fragment was then digested with the Notl restriction endonuclease and ligated into pVWF-1570 digested with Notl. The resulting plasmid containing the naturally occurring VWF coding sequences, the human albumin and linker sequence was named pVWF-1574.

In order to achieve the expression of a fusion protein, several bases had to be removed between VWF and the linker sequence. This was carried out by site-directed mutagenesis according to standard protocols (site-directed mutagenesis kit QuickChange XL, Stratagene, La Jolla, CA, USA) using oligonucleotides We2995 and We2996 (SEQ ID NO 21 and 22 ). The resulting expression plasmid called pVWF-1572 contained the VWF phase coding sequences with that of a 31 amino acid glycine / serine linker and human albumin. The amino acid sequence of rVWF-FP expressed is set forth as SEQ ID No. 25. The amino acid sequence of the human VWF preproprotein is set forth as SEQ ID NO. 24.

Using the plasmids and protocols described above and applying molecular biology techniques known to those skilled in the art (and as described, e.g., in Current Protocols in Molecular Biology, ibid), an expert can prepare other constructs to replace the albumin sequence by another HLEP sequence or the connector sequence by another connector sequence.

Example 8: Purification of VWF and VWF-albumin fusion proteins

Supernatants from cell cultures containing naturally occurring VWF (naturally occurring rVWF) or VWF-albumin fusion protein (rVWF-FP) were filtered sterile through a 0.2 pm filter and dialyzed against a buffer equilibration (EB: 10mM Tris-HCl, CaCl210mM, pH 7.0). This material was then applied to a column of Hectoin Fractogel equilibrated with EB. The column was washed with EB and the VWF proteins were eluted with 500mM NaCl in EB. The elution peak was concentrated and dialyzed against an FB buffer (3 g / L sodium chloride, 20 g / L glycine, 5.5 g / L trisodium citrate dihydrate, pH 7.0). Finally, the material was sterile filtered and frozen in aliquots. When appropriate, additional purification steps were applied comprising anionic and / or cationic exchange chromatography, HIC and SEC.

Example 9: VWF activity and antigen analysis

Samples were analyzed by VWF immunoturbidimetric determination: Ag (OPAB03, Siemens Healthcare Diagnostics, Marburg, Germany) and to determine collagen binding (VWF ELISA: Technozym CBA, Ref. 5450301 with the 5450310 calibration set and the set No. 5450312, Technoclone, Vienna, Austria) as described by the manufacturer.

The VWF: RCo evaluation was carried out using the BC VWF reagent from Siemens Healthcare Diagnostics, Marburg, Germany, according to the manufacturer's description. The international standard of concentrate was used as the main standard preparation to calibrate a standard preparation obtained internally for daily use.

The relationships of the VWF: RCo and VWF: Ag tests are calculated in order to compare this parameter for different constructs evaluated. As shown in Figure 3, the VWF: RCo / VWF: Ag ratio was comparable for naturally occurring rVWF and the rVWF-C-terminal-albumin fusion protein.

For pharmacokinetic analyzes, the VWF antigen was determined by ELISA, the performance of which is known to those skilled in the art. In summary, microplates were incubated with 100 pL per well of the rabbit capture antibody (human anti-VWF IgG, Dako A0082 [Dako, Hamburg, Germany], with a 1: 2000 dilution factor in buffer A [Sigma C3041, Sigma- Aldrich, Munich, Germany]) overnight at room temperature. After washing the plates three times with buffer B (Sigma P3563), each well was incubated with 200 pL of buffer

Claims (22)

5 10 fifteen twenty 25 30 35 40 Four. Five fifty 1. A modified factor VIII or a complex comprising modified factor VIII and unmodified VWF, wherein the modified factor VIII is fused in a C-terminal part of the primary translation polypeptide of factor VIII with the N-terminal part of an albumin. 2. A modified factor VIII or a complex comprising modified factor VIII and unmodified VWF according to claim 1, wherein to. the modified factor VIII has a prolonged functional half-life compared to the functional half-life of factor VIII of natural origin or b. The complex comprising modified factor VIII and unmodified VWF has a prolonged functional half-life compared to the functional half-life of the corresponding complex comprising factor VIII of natural origin and VWF of natural origin. 3. A modified factor VIII or a complex comprising said modified factor VIII or a complex comprising modified factor VIII and unmodified VWF according to claim 2, wherein the modified factor VIII exhibits an increase in the functional half-life of at least one 25% compared to the functional half-life of the corresponding factor VIII of natural origin or a complex comprising said modified factor VIII and unmodified VWF shows an increase in the functional half-life of at least 25% compared to the corresponding FVIII complex of Natural origin and VWF of natural origin. 4. A modified factor VIII or a complex comprising modified factor VIII and unmodified VWF according to claim 1, wherein to. the modified factor VIII has a prolonged half-life of the antigen compared to the half-life of the naturally occurring factor VIII antigen or b. the complex comprising modified factor VIII and unmodified VWF has a prolonged half-life of the antigen compared to the half-life of the corresponding complex antigen comprising factor VIII of natural origin and VWF of natural origin. 5. A modified factor VIII or a complex comprising modified factor VIII and unmodified VWF according to claim 4, wherein the modified factor VIII exhibits an increase in the half-life of the antigen of at least 25% compared to the half-life of the corresponding factor VIII antigen of natural origin or a complex comprising said modified factor VIII and unmodified VWF has an increase in the half-life of the antigen of at least 25% compared to the corresponding complex of FVIII of natural origin and VWF of origin natural. 6. A modified factor VIII or a complex comprising modified FVIII and unmodified VWF according to claim 1, wherein to. Modified factor VIII shows an increase in in vivo recovery compared to in vivo recovery of naturally occurring factor VIII or b. the complex comprising modified factor VIII and unmodified VWF has an increase in in vivo recovery compared to in vivo recovery of the corresponding complex comprising factor VIII of natural origin and VWF of natural origin. 7. A modified factor VIII or a complex comprising modified factor VIII and unmodified VWF according to claim 6, wherein the modified factor VIII exhibits an increase in in vivo recovery of at least 10% compared to the in recovery of the corresponding factor VIII of natural origin or a complex comprising said modified factor VIII and unmodified VWF has an increase in recovery in vivo of at least 10% compared to the corresponding complex of factor VIII of natural origin and VWF of natural origin 8. A modified factor VIII or a complex comprising modified factor VIII and unmodified VWF according to any of the preceding claims, wherein the modified factor VIII has at least 10% of the biological activity of naturally occurring factor VIII or the complex comprising the modified polypeptide or a complex comprising said modified polypeptides has at least 10% of the biological activity of the corresponding complex of naturally occurring FVIII and VWF of natural origin. 9. A recombinant modified FVIII according to any of the preceding claims, wherein said recombinant modified FVIII is secreted from mammalian cells with a higher yield than the naturally occurring FVIII. 10. A polynucleotide or a group of polynucleotides, which encode a modified factor VIII according to any of claims 1-9. 11. A plasmid or vector comprising a polynucleotide according to claim 10, said plasmid or vector being an expression vector or a transfer vector for use in human gene therapy. 12. A host cell comprising a polynucleotide according to claim 10 or a plasmid or vector according to claim 11. 13. A method of producing a modified factor VIII, comprising: (a) culturing host cells according to claim 12 under conditions where the modified factor VIII is expressed; Y 10 (b) optionally recovering modified factor VIII from host cells or culture medium. 14. A pharmaceutical composition comprising a polypeptide or a complex comprising said modified factor VIII according to any of claims 1-9, a polynucleotide according to claim 10, or a plasmid or vector according to claim 11. 15. The use of a polypeptide or a complex comprising said modified factor VII according to any of claims 1-9, a polynucleotide according to claim 10, or a plasmid or vector according to with claim 11, or a host cell according to claim 12 for the preparation of a medicament for the treatment or prevention of a blood clotting disorder. 16. The use according to claim 15, wherein the blood clotting disorder is hemophilia A. 17. The use according to claims 15, wherein the treatment comprises human gene therapy. [mU / mL] Figure 1: Levels of antigen and activity of FVIII of natural origin (457) and of FVIII-C-terminal-albumin fusion polypeptides (1434) image 1 activity / antigen ratio FVIILAg [U / mL] Figure 2: Comparison of the pharmacokinetic parameters of human FVIII: Ag in mice with VWF suppression after i.v. 100 U (FVIII: Ag) / kg of FVIII of natural origin and FVIII -FP 1656 VWF (mean; n = 4 / evaluation point) image2 image3 Figure 3: Proportions of VWF: RCo / VWF: Ag of cell culture supernatants containing naturally occurring rVWF (1570/797), rVWF-FP (1572/797) containing albumin bound to the C-terminal end, or a mixed expression cell culture containing a mixture of naturally occurring rVWF (1570/797) and rVWF-FP (1572/797) transfected with a proportion of 5: 1. Values of approximately 0.8 were obtained in each case, which are close to 1, which corresponds to the theoretical ratio of NHP according to unit definitions 1.20 1.00 OR) 0.80 0.90 0.79 0.76 0.60 0.40 0.00 rVWF of natural origin rVWF-albumin rVWF of natural origin / (1572/797) rVWF- albumin 1570/797) (1570/1572/797) with a proportion of Figure 4: Electrophoresis in agarose-SDS gel of naturally occurring rVWF (1570/797) (B) and rVWF-FP (1572/797), both expressed in HEK cells (A). Bands were detected using either VWF or albumin (ASH) antibodies. rVWF-FP VWF of natural origin rVWF-FP image4 ABA A = rVWF-FP (expressed in the presence of furin) B = VWF of natural origin (expressed in the presence of furina) image5 Figure 5: FC analysis of naturally occurring rVWF and rVWF-FP in rats based on VWF determination: Ag 100 rvWF of natural origin rvWF-FP 200 220 240 100 120 140 160 1B0 Time [minutes]